The emergence of the tetrathionate reductase operon in the Escherichia coli/Shigella pan‐genome

Abstract Escherichia coli pathogenic variants (pathovars) are generally characterized by defined virulence traits and are susceptible to the evolution of hybridized identities due to the considerable plasticity of the E. coli genome. We have isolated a strain from a purified diet intended for research animals that further demonstrates the ability of E. coli to acquire novel genetic elements leading potentially to emergent new pathovars. Utilizing next generation sequencing to obtain a whole genome profile, we report an atypical strain of E. coli, EcoFA807‐17, possessing a tetrathionate reductase (ttr) operon, which enables the utilization of tetrathionate as an electron acceptor, thus facilitating respiration in anaerobic environments such as the mammalian gut. The ttr operon is a potent virulence factor for several enteric pathogens, most prominently Salmonella enterica. However, the presence of chromosomally integrated tetrathionate reductase genes does not appear to have been previously reported in wild‐type E. coli or Shigella. Accordingly, it is possible that the appearance of this virulence factor may signal the evolution of new mechanisms of pathogenicity in E. coli and Shigella and may potentially alter the effectiveness of existing assays using tetrathionate reductase as a unique marker for the detection of Salmonella enterica.

nonhereditary acquisition of different genes of introgressive origin (Bapteste et al., 2012). As a consequence of the genomic acquisition, a variety of strains emerge possessing an array of adaptive or pathogenic traits (Baquero & Tobes, 2013), thereby displaying a remarkable genomic fluidity (Brunder & Karch, 2000;Hazen et al., 2017;Pasqua et al., 2017;Prager et al., 2014). Perhaps a rather dramatic example of this is the major outbreak caused by Escherichia coli of serotype O104:H4 which spread throughout Europe in 2011. This event, regarded as the most lethal of its kind ever reported (Sloup et al., 2016), was caused by an atypical strain that is most similar to enteroaggregative E. coli (EAEC) of serotype O104:H4. This EAEC variant, however, was found to possess a prophage encoding the Shiga toxin, which is characteristic of enterohemorrhagic E. coli (EHEC) strains. This combination of genomic features, uniting virulence factors from both EAEC and EHEC, represents an example of an emergent new pathovar (Navarro-Garcia, 2014) designated as Enteroaggregative Enterohemorrhagic E. coli, or EAHEC. Introgressive descent refers to "the incorporation (usually via hybridization and backcrossing) of alleles from one entity (species) into the gene pool of a second, divergent entity (species)" (Harrison & Larson, 2014). In this report, we demonstrate a further example of this concept with the discovery of a strain possessing genetic elements previously unreported in E. coli.
Our laboratory routinely tests research animal feeds used at our institute for microbial burden and food-borne pathogens such as Salmonella. While screening a purified, high-fat rodent diet, we isolated a novel strain of E. coli designated EcoFA807-17, displaying an indeterminate biochemical profile while conducting an enrichment protocol for the detection of Salmonella enterica. S. enterica shares a morphological resemblance with EcoFA807-17 on Brilliant Green ( Figure 1) and MacConkey Agars, which is also remarkable in the case of Brilliant Green because this selective plate medium tends to inhibit the culture of many strains of E. coli (Moats & Kinner, 1974) including Shigellae (Kristensen, 1925;Moats & Kinner, 1974). After initially characterizing the isolate via multi-locus sequence analysis (MLSA) as E. coli rather than Salmonella, and upon completion of whole genome sequencing (WGS), the isolate appears to possess certain known virulence genes and phenotypes suggesting pathogenic potential, including a functional tetrathionate reductase operon. Tetrathionate respiration, conducted under conditions of anaerobiosis and gut inflammation, serves to enable ttr(+) prokaryotes to outcompete the endogenous intestinal microbiome and is therefore considered a virulence marker in certain enteric pathogens, most notably Salmonella enterica (Winter & Bäumler, 2011). 2 | METHODS AND MATERIALS 2.1 | Isolation and morphologic/biochemical phenotyping of EcoFA807-17 EcoFA807-17 was initially isolated from culture derived from Rappaport-Vassiliadis broth, then subcultured upon MacConkey (MAC) and Brilliant Green (BG) agars. To initially confirm/disprove Salmonella with which the isolate shared a morphological resemblance on both of these two differential agars, cultures were grown on Triple Sugar Iron (TSI) slant media testing for H 2 S production (Binet et al., 2018). Additional metabolic phenotyping was performed on the isolate using the Analytic Profile Index (API) 20E system (bioMérieux, Inc.). To differentiate between E. coli and Shigella, isolates were inoculated into Motility Media with Tetrazolium growth Indicator (Hardy Diagnostics). Isolates were also cultured on Xylose-Lysine-Deoxycholate (XLD) Agar, specifically to confirm the isolate's inability to metabolize Xylose (Silva et al., 1980).

| Antibiotic resistance profile analysis
Testing via the disk diffusion method was conducted to determine the susceptibility of EcoFA807-17 to selected members of classes of antimicrobial agents including the beta-lactam, macrolide, fluoroquinolone, aminoglycoside, sulfonamide, tetracycline, and chloramphenicol families.

| Species identification using multi-locus sequence analysis
Multi-locus sequence analysis (MLSA) was performed as part of the initial species identification process. Total genomic DNA (gDNA) was ac.uk/projects/fastqc/) and no trimming or adaptor removal was indicated. Sequence assemblies and genome annotation were done with Patric 3.5.30 (https://www.patricbrc.org/; (Wattam et al., 2017).
Assembly with this web-based toolkit is done with the SPAdes assembler (Bankevich, Nurk, et al., 2012) and resulted in an assembly of 259 contigs. The genome size is 4,964,044 bp and the genome coverage is 330X. Annotation is based on the RASTtk annotation pipeline (Brettin et al., 2015). The annotation advantage of using Patric is that it contains seven databases related to proteins involved in bacterial pathogenesis, virulence, antibiotic resistance, and drug targets.
Based on our comparison to prokka (Seemann, 2014), a standard prokaryotic annotation tool, Patric provides a much more accurate annotation of draft prokaryotic genomes and allowed us to identify the ttr operon.

| Analysis of genes involved in tetrathionate respiration
Whole genome sequencing identified genes involved in tetrathionate respiration within EcoFA807-17. To confirm their presence, we first performed PCR analysis utilizing the assay previously reported to detect the Salmonella-specific ttr locus (Malorny et al., 2004). In addition, we performed PCR amplification of the individual ttr genes (A, B, C, R, and S) using PCR primers designed (Table 2) Note: The thermal cycling parameters were 95°C for 3 min followed by 35 cycles of 95°C for 15 s for denaturation, 60/62/64°C (60°C -ttrA, ttrC, ttrS, 62C -ttrB, 64C-ttrR) for 30 s for the annealing step, and 72°C for 1 min for extension. A final extension step of 72°C for 5 min was included.  (Casalino et al., 2003;Gomig et al., 2015;Maurelli et al., 1998), although it demonstrates Indole production, a trait long known to be typical of E. coli but much less common with Shigella (Rezwan et al., 2004) and certain other members of Enterobacteriaceae. Its sugar utilization resembles Shigella in being unable to metabolize lactose, sucrose, and xylose (Taylor, 1965). However, EcoFA807-17 differs from Shigella spp. in being robustly motile (Beld & Reubsaet, 2012). In contrast to many pathogens in which wide-spectrum antibiotic resistance is frequently characteristic, EcoFA807-17 was found to be generally susceptible to a broad array of antibiotic agents (Table 4).
When cultured under anaerobic conditions in (Iodine-Iodide) activated tetrathionate broth to simulate an environment of inflammation within the gut, EcoFA807-17 robustly demonstrated the biological activity of its suite of ttr genes with growth similar to the positive control Salmonella and Citrobacter strains.
Conversely, the ttr(−) strain E. coli remained inhibited and failed to grow at the same rate as EcoFA817-17 and the other ttr(+) species when transferred to MacConkey agar ( Figure 2). This result was further emphasized with the successful culture of ttr(−) E. coli on MacConkey agar derived from tetrathionate broth that had not been amended with the iodine-iodide solution as seen in Figure 2.

T A B L E 4 EcoFA807-17 antibiotic analysis
Antibiotic/antibiotic class

Diameters of inhibition zones (mm) MIC results
Ampicillin 10  3.2 | Multi-locus sequence analysis of EcoFA807-17 The housekeeping gene targets in Table 5 were examined via NCBI BLAST for sequence similarity, confirming EcoFA807-17 as a member of the E. coli/Shigella genomospecies.
To further differentiate between E. coli and Shigella, the presence of a ybbW (allantoin permease) allele was affirmed by rtPCR assay and by computational prediction derived from whole genome sequencing.

| Sequencing analysis of ttr elements
To confirm the computational prediction of existing ttr genes, partial amplicons of all five genes were created via PCR and submitted for sequencing (Genewiz, Inc.), then examined for confirmation of protein identification and taxonomic data via the NCBI BLASTX and BLASTN Bioinformatics search tools, respectively. Despite the significant divergence in nucleotide identity between the EcoFA807-17 and C. freundii homologs, we were consistently successful in amplifying most of the ttr elements of both species with primers specifically targeting EcoFA807-17 ttr homologs, except for the ttrB structural gene of C. freundii (Figure 3, Lane 15), using stringent annealing temperatures consistent with primer melting temperatures (Tm).
By contrast, we were unable to amplify Salmonella ttr homologs, nor did we detect the presence of ttr elements in EcoFA807-17 via a Salmonella-specific PCR assay (Malorny et al., 2004), thus providing additional support for the possibility that EcoFA807-17 has acquired its ttr elements from a Citrobacter lineage.  Table 6. Only approximately 0.7% of these genomes contained all five genes comprising the ttr operon, whereas the lacZ gene common to coliform bacteria (Molina et al., 2015), and the ybbW gene, diagnostic of E. coli, were present in 97.8 and 96.3 of this E. coli genome data set, respectively.
To confirm that these genes were present in a true operon configuration, EcoFA807-17 and a sample of 27 complete E. coli genomes of those strains in Table 7 predicted to have a complete ttr operon were re-annotated using the Patric annotation pipeline described in Methods. Where the assembly annotation indicated a plasmid localization of the ttr operon is also noted in Table 7.
A similar strategy was used to search available NCBI Shigella genomes, as of 5 May 2020. One S. sonnei strain was found to possess the ttr operon, as were just three other additional Shigella strains including one strain of S. flexneri and two Shigella strains of undetermined species. No S. dysenteriae or S. boydii genomes were found to contain a ttr operon.
Shigella strains containing a complete ttr operon are listed in differentiation between E. coli and Shigella. EcoFA807-17 resembles Shigella in some respects rather than E. coli, most strikingly by its inability to ferment xylose (Altwegg et al., 1996;de Boer, 1998;Taylor, 1965). Further complicating differentiation between E. coli and the obligate pathogen Shigella, the close phylogenetic relationship between the two organisms thwarts the use of household genes typically used in multi-locus sequence analysis for identification at the species level. Those MLSA targets used for the initial genetic examination include rrn (ribosomal RNA or 16S), gyrB, and rpoB.
The sequencing and attempted species identification via these genes deserve further comment. Long considered the "gold standard" for taxonomic classification (Case et al., 2007) of bacterial species, the rrn (16S) gene is effective for taxonomic determination at the genus level and sometimes at the species level as well, but is unable to decisively discriminate between E. coli and Shigella (Christensen et al., 1998;Devanga Ragupathi et al., 2018), although its employment was initially instrumental in confirming that EcoFA807-17 was not Salmonella. This was not surprising given that Escherichia and Shigella, while deemed The PCR amplicons of the various ttr locus elements were resolved on a 2.0% agarose gel. The lanes are as follows: Note: Trace bands in Lanes 16, 20, and 21 were subjected to additional PCR and determined via sequencing and BLAST analysis to be nonspecific amplification.
separate taxa for purposes of clinical distinction (Farmer et al., 1985;Rezwan et al., 2004), are genetically the same species (Beld & Reubsaet, 2012;Fukushima et al., 2002). They are therefore too closely related to be reliably differentiated by the highly conserved and frequently multi-copy ribosomal RNA genes (Devanga Ragupathi et al., 2018). Similarly, the gyrB and rpoB household genes which are generally more discriminatory than rrn (16S) at the species and even sub-species level, were determined nonetheless to be insufficiently useful to differentiate between E. coli and Shigella as demonstrated by inconclusive results (Table 5) obtained from NCBI data of the housekeeping gene sequences submitted. By extension, they are also deemed wholly unsuitable for serovar/biovar determination (Adékambi et al., 2009).
Accordingly, a real-time PCR assay described by Walker et al., (Walker et al., 2017) was utilized to enable a decisive confirmation of EcoFA807-17 as E. coli. The principle underlying this method is that the ybbW gene, which codes for allantoin permease, has been regarded as universally inclusive and exclusive to E. coli (but not Shigella) and subsequent testing confirmed that EcoFA807-17 is ybbW (+). We observe, however, that some data from this study suggests that ybbW may not be as exclusive to E. coli as originally thought. Specifically, we note that a number of strains of S. sonnei and S. flexneri found within NCBI data (Table 8, right column) appear to possess the ybbW gene, including ttr(+) S. sonnei strain ECSW + 10 (GCA_002248745.1). In the case of the latter, while conceivable that ECSW + 10 is an E. coli (EIEC) strain originally misclassified as S. sonnei, this is a less likely explanation as further examination of the genome of S. sonnei strain ECSW + 10 reveals the predicted presence of a pINV type B invasion plasmid. The latter is characteristic of S. sonnei while closely related EIEC strains and certain other Shigellae tend to possess pINV Type A invasion plasmids (Lan et al., 2001. The unexpected presence of ybbW as well as a ttr operon within S. sonnei strain ECSW + 10 serves to further emphasize the dynamic mutability of the E. coli/Shigella pan-genome. Nevertheless, the presence of the ybbW gene is observed to be uncommon among the several thousand Shigella genomes deposited at NCBI. For that reason and because the Shigellae universally possess pINV invasion plasmids and lack motility (Beld & Reubsaet, 2012), we conclude that EcoFA807-17, which lacks the former and is highly motile, is properly classified as a strain of E. coli.

| Characterization of the ttr operon of EcoFA807-17
Initially (in 2018), the ttr operon sequences of EcoFA807-17 were determined via NCBI BLASTN to be most closely related to Citrobacter freundii homologs with shared nucleotide identity ranging from 85% to 90% as described from NCBI data. Reinforcing this data, we found that our ttr PCR primers, designed from sequences predicted of EcoFA807-17, not only successfully PCR-amplified and confirmed the computationally indicated existence of these genes, but also PCR-amplified four out of the five counterparts found in C. freundii (see Figure 3), despite the significant degree of deviation in analysis, the ttr operon locus is not on either of these two plasmids and thus is likely chromosomal. This is consistent with the fact that of the ttr(+) strains listed in Table 7, relatively few appear to possess plasmid-borne ttr elements. To address the question of whether ttr elements within EcoFA807-17 were biologically active, the strain was cultured anaerobically in tetrathionate broth as previously described.

Simulation of the in vivo infection and inflammation process is
achieved with the addition of the Iodine-iodide solution to the broth. T A B L E 6 Incidence of ttr genes relative to common household genes (ybbW, lacZ) in E. coli genomes in NCBI

| Significance of ttr operon acquisition
There is a relative paucity of literature before this century concerning tetrathionate respiration in the Enterobacteriaceae family since the phenomenon was first described by Pollock and co-workers in the early 1940s (Kapralek, 1972). However, studies conducted in the 1970s by Kapralek and Rezbova with their work confined to the genus Citrobacter, revealed that tetrathionate respiration enhanced specific growth rate, raised growth yield, and enabled growth on non-fermentable carbon sources. By 1999, Hensel and colleagues had identified and mapped the structure and functions of the operon responsible for tetrathionate metabolism in Salmonella (Hensel et al., 1999). Even then, the role of tetrathionate reductase as a virulence factor was not yet established as Hensel surmised that ttr metabolism was a survival trait utilized by Salmonella in the external environment rather than within eukaryotic hosts. This assumption is understandable, given that there were no known sources of tetrathionate in the mammalian host (Winter et al., 2010 (Ribeiro et al., 2017), some Citrobacter are recognized as opportunistic pathogens such as the ttr(+) species C. freundii and C. amalonaticus (Lipsky et al., 1980). Nevertheless and apart from Salmonella, the literature is sparse concerning the role of tetrathionate respiration as a virulence factor in other bacteria (Hensel et al., 1999) including Citrobacter. and in vivo." (Anderson et al., 2017) As tetrathionate respiration also serves to promote those organisms possessing that capability in interbacterial competition, EcoFA807-17 may potentially have a formidable advantage over endogenous microbiota.
Our data (Table 7) suggests that the operon is gradually proliferating through the past decade in Escherichia coli, though it is present in less than 1% of genomes deposited at NCBI. In contrast, its presence in Shigella (Table 8)

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
In addition to the sequence submission to the Short Read Archive (accession number SRR14216030), this Whole Genome Shotgun project has also been deposited at DDBJ/ENA/GenBank under the accession JAGRQF000000000: The version described in this paper is version JAGRQF010000000: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA 721428.

ETHICS STATEMENT
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